Kromatografi Cair Kinerja Tinggi (KCKT)

High Performance Liquid Chromatography (HPLC)

Alasan dikembangkannya HPLC:
 Sebagian besar (85 %) senyawa yang ada tidak cukup volatil atau tidak cukup
stabil untuk dianalisis dengan GC, sehingga diperlukan reaksi derivatisasi
dulu untuk dapat dianalisis atau bahkan sama sekali tidak dapat dianalisis
dengan GC.

 Pendekatan tradisional pada kromatografi cair kolom terbuka memerlukan

solven dan sampel yang sangat banyak. Aliran fasa gerak sangat lambat
karena bergantung gravitasi, sehingga pemisahan berlangsung dalam order
jam. Fraksi yang terkumpul dari pemisahan harus dianalisis secara terpisah
sehingga memperlama waktu analisis.

 Dengan HPLC pemisahan dan identifikasi dilakukan bersama-sama dalam

order menit.
 High Performance Liquid Chromatography

HPLC is characterized by the use of high pressure to push a

mobile phase solution through a column of stationary phase
allowing separation of complex mixtures with high resolution.
 Chromatography Schematic
MOBILE PHASE LIQUID

Liquid-Liquid Liquid-Solid
FORMAT Chromatography Chromatography
(Partition) (Adsorption)

STATIONARY Solid
Liquid
PHASE

Normal Phase Normal Phase Reverse Phase

Reverse Phase

Mobile Phase - Mobile Phase -

Nonpolar Polar
Stationary phase - Stationary phase -
Polar Nonpolar
 COMPOSITION OF A LIQUID
CHROMATOGRAPH SYSTEM
 Solvent
 Solvent Delivery System (Pump)
 Injector
 Sample
 Column
 Detectors (Diode Array)
 Waste Collector
 Recorder (Data Collection)
 Instrumentation

Gradient
Controller
•
Pump Column
Detector
Injector
Mobile Phases
Chromatography II: HPLC
Hewlett-Packard
Series 1100 HPLC

water methanol caffeine

(very polar) (polar) (fairly nonpolar)
 Chromatography II: HPLC
HPLC Column

solvent
(mobile phase) to detector
and
sample
wax coated beads

nonpolar
stationary phase

microscopic view of bead

3 m
 HPLC - Modes
 Normal Phase.
- Polar stationary phase and non-polar
solvent.

• Reverse Phase.
- Non-polar stationary phase and a polar
solvent.
Common Reverse Phase Solvents
 Methanol CH3OH
• Acetonitrile CH3CN

• Tetrahydrofuran

• Water H2O
 HPLC Chromatograph injectors
 The function of the injector is to place the sample into the
high-pressure flow in as narrow volume as possible so that
the sample enters the column as a homogeneous, low-
volume plug. To minimize spreading of the injected
volume during transport to the column, the shortest
possible length of tubing should be used from the injector
to the column.

 When an injection is started, an air actuator rotates the

valve: solvent goes directly to the column; and the injector
needle is connected to the syringe. The air pressure lifts
the needle and the vial is moved into position beneath the
needle. Then, the needle is lowered to the vial.
 Injektor

Tempat memasukkan sampel dengan volume loop tertentu. Mis:

100, 50, 20 dan 10 L
 Sample Injection System
Used to introduce
small samples
(0.001 to 0.5 mL)
into the carrier
stream under
high pressure
 HPLC columns
 The column is one of the
most important components
of the HPLC chromatograph
because the separation of the
sample components is
achieved when those
components pass through the
column. The High
performance liquid
chromatography apparatus is
made out of stainless steel
tubes with a diameter of 3 to
5mm and a length ranging
from 10 to 30cm.
 Normally, columns are filled with
silica gel because its particle
shape, surface properties, and
pore structure help to get a good
separation. Silica is wetted by
nearly every potential mobile
phase, is inert to most compounds
and has a high surface activity
which can be modified easily
with water and other agents.
Silica can be used to separate a
wide variety of chemical
compounds, and its
chromatographic behavior is
generally predictable and
reproducible.
Picture of an HPLC column
 Detektor
 Detektor Refraktometer Differensial: mengukur perubahan indeks refraksi
eluen yang disebabkan oleh keberadaan solut dalam eluen yang keluar dari
kolom. Merupakan detektor universal. Sensitivitas: 10-5 – 10-6 g/mL (10-1
ppm)
 Detektor Ultraviolet-Visible: sensitivitas sampai 0,01 ppm, tidak sensitif thd
perubahan temperatur, tidak mahal dan dapat merespon sejumlah besar
senyawa-senyawa organik. Kira-kira 80 % pengukuran memakai HPLC
menggunakan detektor ini.
 Detektor Fluoresensi: untuk senyawa-senyawa yang dapat berfluoresensi,
dapat lebih sensitif dari UV-Visible.
 Detektor Amperometrik: untuk senyawa-senyawa yang elektroaktif. Tidak
sesensitif detektor UV. Detektor ini banyak dipakai pada analisis biokimia,
misalnya, pemisahan dan deteksi konsentrasi runut katekolamin dalam otak
 Varian HPLC System
9060 Polychrom Computer
(Diode Array) Detector Workstation

9050 Variable
9010 Solvent UV/Vis Detector
HPLC Solvent Delivery System
Reservoirs

Rheodyne
Injector

Keep an eye on
HPLC these 4 screens!
Column
Varian Solvent Delivery System
 %A %B %C Flow Rate Pressure to column
{H2O} {MeOH} (mL/min) (atmos.)
load

Ready
inject

Rheodyne
Injector
Varian 9010 Solvent Delivery System to injector

through pump

Column
through C
pulse
dampener
A B

from solvent to
Ternary Pump reservoir detector
Variable UV/Vis Detector
ABS AUFS  RunTime EndTime
0.001 2.000 238 0.00 min 10.0 min

Ready
 Ready
UV Spectrum
UV Spectrum {shows full UV abs.}
UVmax
UVmax

Chromatogram

ABS.
Reset
Wavelength

Rt Rt

ABS.
Time

Chromatogram
{shows peaks, Rt}

Varian 9060
Polychrom Detector
Chromatography II: HPLC
Hewlett-Packard
Series 1100 HPLC

solvent

pump

injector

column

detector
Chromatography II: HPLC
HPLC Column
Chromatography II: HPLC
HPLC Pump
Chromatography II: HPLC
HPLC Autosampler and Injector
Chromatography II: HPLC

HPLC Detector
UV/Visible Spectrophotometer
Chromatography II: HPLC

HPLC Detector
UV/Visible Spectrophotometer
Chromatography II: HPLC
• Procedure
 Use a 10 mL volumetric pipet to add 10.00 mL of soft drink to 10.00 mL of
deionized water.

 Mix thoroughly and half fill a HPLC vial with your sample. Label the vial with your
name and the name of the soft drink.
 HPLC Chromatograms Approximation
of peak area by
triangulation
Absorbance 

Peak A Peak B

height

0 1 2 3 4 5 6 7
Time (minutes) base

Rt = 3.0 min. Rt = 5.2 min.

faster moving slower moving base x height
Area =
less retained more retained 2
 Chromatography Stationary Phases

Silica Gel Derivatized Silica Gel

O O O O O O
| | | | | |
OSiOSiOSiOH OSiOSiOSiOR Where R = C18H37
| | | | | |
O O O O O O hydrocarbon chain
| | | | | | (octadecylsilyl deriv.
OSiOSiOSiOH OSiOSiOSiOR silica or “C18”)
| | | | | |
O O O O O O

bulk (SiO2)x surface bulk (SiO2)x surface

relatively polar surface relatively nonpolar surface

“normal phase” “reversed phase”
 Normal vs. Reversed Phase Chromatography
Normal Phase Reversed Phase
Stationary phase Polar (silica gel) Non-polar (C18)
Non-polar Polar
Mobile phase
(organic solvents) (aqueous/organic)
Sample movement Non-polar fastest Polar fastest
Different polarities Different
Separation based on
(functionality) hydrocarbon content